THE REGULATION OF DIFFERENTIATION FROM STEM CELLS TO THE FIRST SYNAPSE IN THE MAMMALIAN CNS. Research in this group is focused on two aspects of the development of the central nervous system: 1. The identification and characterization of stem and progenitor cells. 2. Regulation of neuron-target interaction. Multipotential cells with similar responses to extracellular differentiation signals can be found in both the fetal and adult central nervous system. The identification of a stem cell in the mammalian CNS raises the question of the origin of this cell type and how other cell types are derived from it. In recent experiments we show that the CNS stem cells can give rise directly to peripheral nervous system stem cells. This is true even when the CNS cells are derived from anterior structures that do not normally form neural crest. These results show that cells throughout the nervous system share common differentiation mechanisms. A clear limitation of these experiments is that they analyze cell function in tissue culture. The use of grafting to study cellular properties provides data on the responses of cells in vivo. Recent grafting experiments from LMB provide strong evidence for unexpected plasticity in the differentiation of CNS precursor cells. In these experiments, cells were grafted as a suspension into the developing brain. Donor cells enter the tissue and differentiate into neurons and glia which are indistinguishable from host cells at the site where the cells implant. This is true even when the donor cells are derived from a distinct region of the nervous system. The incorporation of neurons and glia into the structure of the host brain suggests that local extracellular signals acting on a common stem controls neuronal differentiation. These in vitro and in vivo experiments stress that cells in the developing nervous system share developmental potential. Precursor cells in the developing and adult nervous system express a specific intermediate filament protein (nestin). The regulation of nestin expression has been studied in transgenic mice to identify transcriptional mechanisms that control, stem cell specific gene expression. A POU site and a hormone response element define stem cell specific gene expression in the nestin gene and in a second protein with a similar pattern of expression to nestin. The parallels between the regulatory elements in these two genes suggest that the POU proteins play a central role in establishing the common properties shared by CNS stem cells. When stem cells differentiate they rapidly generate neurons. Neuronal differentiation is a complex process that takes several days to accomplish. A model system for our work has been the rodent telencephalon and particularly the action of neurotrophins on hippocampal neurons. We have established that neurotrophins act at both the earliest stages of differentiation to neuronal subtypes and also later to control the activation of glutamatergic and GABA-ergic synapses. These findings are particularly striking as NT3 and BDNF have distinct effects on the activation of glutamatergic and GABA-ergic synapses. The action of neurotrophins to regulate neuron-target interactions has played an important conceptual role in neuroscience. The availability of knockout mice has made a significant contribution to understanding neurotrophin signaling in the PNS but , in spite of a great deal of work, the role of neurotrophins in the CNS is still not clear. The in vitro assays we have established will assist in defining the roles of neurotrophins in regulating synaptic interactions between neurons. In another approach to the analysis of early synapses, transgenic mice have been generated expressing NMDA receptor subunits that are normally only transiently expressed in developing neurons. In the transgenics, the function of the NR2D subunit of the NMDA receptor was analyzed by electrophysiology in hippocampal slices. These experiments show that the role of the first NMDA receptors to be expressed in the hippocampus is very different from the well characterized functions of NMDA receptors in later synapses. In spite of severe deficits in both long term potentiation and depression, spatial learning was normal. These in vivo experiments suggest that the NMDA receptor can not use frequency dependent modulation of synaptic properties to control the earliest steps in synaptic activation. The LMB was established in 1993 as a new group using multidisciplinary approaches to study stem cell differentiation and synaptic activation in the CNS. The major achievements of the LMB have been to establish experimental systems to define these two key events in CNS development. This work provides a strong foundation for further analysis of extracellular signals and intracellular pathways that regulate cell type and circuit formation in different regions of the central nervous system. In addition to the grafting experiments that define the developmental properties of CNS cells, we have used grafting tools to establish new models for the treatment of multiple sclerosis, Parkinson?s and Huntington?s disease. The results of these experiments have direct implications for cell therapies in neurologic disease but a moment?s reflection will reveal a deeper significance. The startling new access to the mechanisms that control cellular differentiation and synaptic circuitry in the CNS will also provide new pharmacological tools to treat a wide range of CNS diseases.